Elevated blood pressure, heart rate and body temperature in mice lacking the XL alpha s protein of the Gnas locus is due to increased sympathetic tone

NEW FINDINGS: What is the central question of this study? Previously, we showed that Gnasxl knock-out mice are lean and hypermetabolic, with increased sympathetic stimulation of adipose tissue. Do these mice also display elevated sympathetic cardiovascular tone? Is the brain glucagon-like peptide-1 system involved? What is the main finding and its importance? Gnasxl knock-outs have increased blood pressure, heart rate and body temperature. Heart rate variability analysis suggests an elevated sympathetic tone. The sympatholytic reserpine had stronger effects on blood pressure, heart rate and heart rate variability in knock-out compared with wild-type mice. Stimulation of the glucagon-like peptide-1 system inhibited parasympathetic tone to a similar extent in both genotypes, with a stronger associated increase in heart rate in knock-outs. Deficiency of Gnasxl increases sympathetic cardiovascular tone. Imbalances of energy homeostasis are often associated with cardiovascular complications. Previous work has shown that Gnasxl-deficient mice have a lean and hypermetabolic phenotype, with increased sympathetic stimulation of adipose tissue. The Gnasxl transcript from the imprinted Gnas locus encodes the trimeric G-protein subunit XLαs, which is expressed in brain regions that regulate energy homeostasis and sympathetic nervous system (SNS) activity. To determine whether Gnasxl knock-out (KO) mice display additional SNS-related phenotypes, we have now investigated the cardiovascular system. The Gnasxl KO mice were ∼20 mmHg hypertensive in comparison to wild-type (WT) littermates (P≤ 0.05) and hypersensitive to the sympatholytic drug reserpine. Using telemetry, we detected an increased waking heart rate in conscious KOs (630 ± 10 versus 584 ± 12 beats min(−1), KO versus WT, P≤ 0.05). Body temperature was also elevated (38.1 ± 0.3 versus 36.9 ± 0.4°C, KO versus WT, P≤ 0.05). To investigate autonomic nervous system influences, we used heart rate variability analyses. We empirically defined frequency power bands using atropine and reserpine and verified high-frequency (HF) power and low-frequency (LF) LF/HF power ratio to be indicators of parasympathetic and sympathetic activity, respectively. The LF/HF power ratio was greater in KOs and more sensitive to reserpine than in WTs, consistent with elevated SNS activity. In contrast, atropine and exendin-4, a centrally acting agonist of the glucagon-like peptide-1 receptor, which influences cardiovascular physiology and metabolism, reduced HF power equally in both genotypes. This was associated with a greater increase in heart rate in KOs. Mild stress had a blunted effect on the LF/HF ratio in KOs consistent with elevated basal sympathetic activity. We conclude that XLαs is required for the inhibition of sympathetic outflow towards cardiovascular and metabolically relevant tissues

Ion Channels in the Paraventricular Hypothalamic Nucleus (PVN); Emerging Diversity and Functional Roles

The paraventricular nucleus of the hypothalamus (PVN) is critical for the regulation of homeostatic function. Although also important for endocrine regulation, it has been referred to as the “autonomic master controller.” The emerging consensus is that the PVN is a multifunctional nucleus, with autonomic roles including (but not limited to) coordination of cardiovascular, thermoregulatory, metabolic, circadian and stress responses. However, the cellular mechanisms underlying these multifunctional roles remain poorly understood. Neurones from the PVN project to and can alter the function of sympathetic control regions in the medulla and spinal cord. Dysfunction of sympathetic pre-autonomic neurones (typically hyperactivity) is linked to several diseases including hypertension and heart failure and targeting this region with specific pharmacological or biological agents is a promising area of medical research. However, to facilitate future medical exploitation of the PVN, more detailed models of its neuronal control are required; populated by a greater compliment of constituent ion channels. Whilst the cytoarchitecture, projections and neurotransmitters present in the PVN are reasonably well documented, there have been fewer studies on the expression and interplay of ion channels. In this review we bring together an up to date analysis of PVN ion channel studies and discuss how these channels may interact to control, in particular, the activity of the sympathetic system

The depressor response to intracerebroventricular hypotonic saline is sensitive to TRPV4 antagonist RN1734

Several reports have shown that the periventricular region of the brain, including the paraventricular nucleus (PVN), is critical to sensing and responding to changes in plasma osmolality. Further studies also implicate the transient receptor potential ion channel, type V4 (TRPV4) channel in this homeostatic behaviour. In previous work we have shown that TRPV4 ion channels couple to calcium-activated potassium channels in the PVN to decrease action potential firing frequency in response to hypotonicity. In the present study we investigated whether, similarly, intracerebroventricular (ICV) application of hypotonic solutions modulated cardiovascular parameters, and if so whether this was sensitive to a TRPV4 channel inhibitor. We found that ICV injection of 270mOsmol artificial cerebrospinal fluid (ACSF) decreased mean blood pressure, but not heart rate, compared to naïve mice or mice injected with 300mOsmol ACSF. This effect was abolished by treatment with the TRPV4 inhibitor RN1734. These data suggest that periventricular targets within the brain are capable of generating depressor action in response to TRPV4 ion channel activation. Potentially, in the future, the TRPV4 channel, or the TRPV4–KCa coupling mechanism, may serve as a therapeutic target for treatment of cardiovascular disease

An in vitro model of skeletal muscle volume regulation.

Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human skeletal muscle cell line TE671RD. In this study we investigated whether hypertonic challenge of the human skeletal muscle cell line TE671RD triggered cell death or evoked a cell volume recovery response.The cellular volume of TE671RD cells was calculated from the 2D surface area. Cell death was assessed by both the trypan blue live/dead assay and the TUNEL assay.Medium osmolality was increased by addition of up to 200 mM sucrose. Addition of 200 mM sucrose resulted in mean cell shrinkage of 44±1% after 30 mins. At later time points (2 and 4 hrs) two separate cell subpopulations with differing mean cell volume became apparent. The first subpopulation (15±2% of the total cell number) continued to shrink whereas the second subpopulation had an increased cell volume. Cell death was observed in a small proportion of cells (approximately 6-8%).We have established that a substantial proportion of TE671RD cells respond to hypertonic challenge with RVI, but that these cells are resistant to hypertonicity triggered cell death

Temperature modulates PVN pre-sympathetic neurones via transient receptor potential ion channels

The paraventricular nucleus (PVN) of the hypothalamus plays a vital role in maintaining homeostasis and modulates cardiovascular function via autonomic pre-sympathetic neurones. We have previously shown that coupling between transient receptor potential cation channel subfamily V Member 4 (Trpv4) and small-conductance calcium-activated potassium channels (SK) in the PVN facilitate osmosensing, but since TRP channels are also thermosensitive, in this report we investigated the temperature sensitivity of these neurones.Methods: TRP channel mRNA was quantified from mouse PVN with RT-PCR and thermosensitivity of Trpv4-like PVN neuronal ion channels characterised with cell-attached patch-clamp electrophysiology. Following recovery of temperature-sensitive single-channel kinetic schema, we constructed a predictive stochastic mathematical model of these neurones and validated this with electrophysiological recordings of action current frequency.Results: 7 thermosensitive TRP channel genes were found in PVN punches. Trpv4 was the most abundant of these and was identified at the single channel level on PVN neurones. We investigated the thermosensitivity of these Trpv4-like channels; open probability (Po) markedly decreased when temperature was decreased, mediated by a decrease in mean open dwell times. Our neuronal model predicted that PVN spontaneous action current frequency (ACf) would increase as temperature is decreased and in our electrophysiological experiments, we found that ACf from PVN neurones was significantly higher at lower temperatures. The broad-spectrum channel blocker gadolinium (100 µM), was used to block the warm-activated, Ca2+-permeable Trpv4 channels. In the presence of gadolinium (100 µM), the temperature effect was largely retained. Using econazole (10 µM), a blocker of Trpm2, we found there were significant increases in overall ACf and the temperature effect was inhibited.Conclusion: Trpv4, the abundantly transcribed thermosensitive TRP channel gene in the PVN appears to contribute to intrinsic thermosensitive properties of PVN neurones. At physiological temperatures (37°C), we observed relatively low ACf primarily due to the activity of Trpm2 channels, whereas at room temperature, where most of the previous characterisation of PVN neuronal activity has been performed, ACf is much higher, and appears to be predominately due to reduced Trpv4 activity. This work gives insight into the fundamental mechanisms by which the body decodes temperature signals and maintains homeostasis.</jats:p

Volume of TE671RD following 30 min of increased osmolality.

<p><b>(A)</b> Cells measured in 300mOsm/Kg H₂O (no sucrose), <b>(B)</b> with 350 mOsm/Kg H₂O (50 mM sucrose), <b>(C)</b> with 400 mOsm/Kg H₂O (100mM sucrose) <b>(D)</b> with 450 mOsm/Kg H₂O (150mM sucrose), <b>(E)</b> with 500 mOsm/Kg H₂O (200mM sucrose). For (A) to (E) smooth lines are Gaussian fits. <b>(F)</b> Normalised volume against sucrose concentration. The smooth line is drawn to that of ideal osmotic behaviour: <i>Vol</i>/<i>Vol</i>₀ = <i>V</i>₀ * <i>osm</i>₀/<i>osm</i>_{<i>sucrose</i>}. Where <i>Vol</i>_<i>o</i> and <i>Vol</i> are the initial volume and the volume attained by the cells in the sucrose containing solution (measured at 30mins). <i>osm</i>_<i>0</i> is the initial osmolality of the solution (I.e., DMEM alone, 300mOsm/Kg H₂O) and <i>osm</i>_{<i>sucrose</i>} is the osmolality once sucrose had been added (i.e., 300mOsm/Kg H₂O + [sucrose]).</p

Cell viability assays.

<p><b>(A)</b> Example of the live/dead trypan blue exclusion assay. Cells are shown in control and then during 0.5h-4h incubation in hypertonic media with 200 mM/Kg H₂O sucrose (from left to right). <b>(B)</b> Quantification of the live/dead trypan blue exclusion assay with time. Cell death with 200mM sucrose at 2hrs and 4hrs was statistically increased from control (p<0.05, Mann-Whitney), but there was no statistical difference between 2hrs and 4hrs. <b>(C)</b> Fluorescent images of TE671RD cells stained by DAPI (blue) and TACS 2 TdT-fluor “Apoptosis Detection Kit” (green) obtained using Nikon Eclipse microscope. Fluorescent microscopy showed the presence of DNA fragmentation after 4 h of 200 mM sucrose incubation. 6±1% (n = 5) of sucrose-treated cells demonstrated a bright green nucleus with condensed chromatin. The TUNEL apoptosis assay was conducted at 4hrs.</p